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author:("Xu, hadong")
1.  Two novel Brugada syndrome-associated mutations increase KV4.3 membrane expression and function 
The human cardiac fast transient outward K+ channel is composed of the KV4.3 α subunit encoded by KCND3 and the K+ channel-interacting protein 2 (KChIP2) β subunit, and determines the early repolarization of the action potential (AP). Two human mutations (G600R and L450F) in KV4.3 are associated with Brugada syndrome and they increase the KV4.3/KChIP2-encoded fast transient outward K+ current (Ito,f) and cause the stable loss of the AP dome. However, the detailed mechanisms underlying the gain of Ito,f function by these two mutations are largely unknown. The experiments in the present study were undertaken to investigate the effect of these mutations and the underlying mechanism. Whole cell patch-clamp recording was performed in HEK-293 cells expressing KV4.3-wild-type (WT) and KV4.3 mutants with KChIP2. The two individual mutant-encoded currents were significantly increased but the kinetics of the channels affected by the two mutations were different. The two mutations slowed KV4.3/KChIP2-encoded channel inactivation; they did not increase the recovery from the KV4.3/KChIP2-encoded channel inactivation. Western blotting showed that total KV4.3 protein was significantly augmented in HEK-293 cells expressing the two individual mutants with KChIP2. Furthermore, immunofluorescence confocal microscopy demonstrated that the KV4.3 channel protein was expressed more in the cell membrane compared to the cytoplasm in cells that expressed individual mutants with KChIP2. Also, KChIP2 increased the amount of channel protein in the cell membrane of KV4.3 mutants significantly more than KV4.3-WT. Reverse transcription-polymerase chain reaction showed that KV4.3 mRNA was not significantly changed by individual mutations in the presence of KChIP2. Taken together, the present study revealed that the mutations cause a gain-of-function of KV4.3/KChIP2-encoded channels by increasing membrane protein expression and slowing channel inactivation.
PMCID: PMC4494594  PMID: 26016905
Brugada syndrome; KV4.3 mutants; KV4.3 functional expression
2.  Malignant melanoma mimicking giant cell variant of glioblastoma multiforme: a case report and review of literature 
We present a case of metastatic malignant melanoma in a patient initially diagnosed with glioblastoma multiforme, giant cell variant. A forty year old female presented to our institution for a re-resection of a recurrent right parietal lobe mass, presumed to be recurrent glioblastoma multiforme. PET scan during preoperative evaluation revealed a 3 cm left lower lobe lung mass. Metastatic glioblastoma to lung was considered in the differential diagnosis. Resection of the brain mass revealed a highly pleomorphic giant and spindle cell lesion with an immunophenotype strongly supportive of melanoma. Immunostains for melanocytic markers were subsequently performed on the lung biopsy specimen, and demonstrated diffuse staining of the atypical cells, supporting the diagnosis of malignant melanoma in the lung. This case demonstrates the importance of considering melanoma in the differential in any tumor with high grade features.
PMCID: PMC4503192  PMID: 26191321
Melanoma; giant cell variant glioblastoma multiforme; pleormorphic lung carcinoma
3.  IL-13 Induces YY1 through the AKT Pathway in Lung Fibroblasts 
PLoS ONE  2015;10(3):e0119039.
A key feature of lung fibrosis is the accumulation of myofibroblasts. Interleukin 13 (IL-13) is a pro-fibrotic mediator that directly and indirectly influences the activation of myofibroblasts. Transforming growth factor beta (TGF-β) promotes the differentiation of fibroblasts into myofibroblasts, and can be regulated by IL-13. However, IL-13’s downstream signaling pathways are not completely understood. We previously reported that the transcription factor Yin Yang 1 (YY1) is upregulated in fibroblasts treated with TGF-β and in the lungs of mice and patients with pulmonary fibrosis. Moreover, YY1 directly regulates collagen and alpha smooth muscle actin (α-SMA) expression in fibroblasts. However, it is not known if IL-13 regulates fibroblast activation through YY1 expression. We hypothesize that IL-13 up-regulates YY1 expression through regulation of AKT activation, leading to fibroblast activation. In this study we found that YY1 was upregulated by IL-13 in lung fibroblasts in a dose- and time-dependent manner, resulting in increased α-SMA. Conversely, knockdown of YY1 blocked IL-13-induced α-SMA expression in fibroblasts. Furthermore, AKT phosphorylation was increased in fibroblasts treated with IL-13, and AKT overexpression upregulated YY1, whereas blockade of AKT phosphorylation suppressed the induction of YY1 by IL-13 in vitro. In vivo YY1 was upregulated in fibrotic lungs from CC10-IL-13 transgenic mice compared to that from wild-type littermates, which was associated with increased AKT phosphorylation. Taken together, these findings demonstrate that IL-13 is a potent stimulator and activator of fibroblasts, at least in part, through AKT-mediated YY1 activation.
PMCID: PMC4361578  PMID: 25775215
4.  Evaluation of 18F-fluorodeoxyglucose uptake in enlarged mediastinal lymph nodes in patients with lung cancer 
Accurate lymph nodal staging of lung cancer is critical for determining the treatment options. With the help of 18F-fluorodeoxyglucose positron emission tomography/computer tomography (18F-FDG-PET/CT), the clinician can rule out/in the regional lymph nodes positive for metastasis in the patients with lung cancer in a majority of cases. However, a small proportion of cases with false positivity of metastasis have been reported. Transbronchial needle aspirations and mediastinoscopic biopsies are still necessary to determine whether enlarged hypermetabolic mediastinal lymph nodes are positive for lung cancer metastasis. Here we report three intricate cases showing hypermetabolic activity in the mediastinal lymph nodes in the patients with pathologically diagnosed lung cancer on PET/CT. The first patient had squamous cell carcinoma in the left upper lobe of the lung with surrounding necrotizing granulomas and concurrent with silicosis and granulomatous inflammation in the lymph nodes; the second presented with symptoms of viral pneumonia, which was pathologically diagnosed as a lung adenocarcinoma, stage IA, concurrent with sarcoidosis involving the lymph nodes; the last case was diagnosed as squamous cell carcinoma in the right upper lobe of the lung, but lymph nodes showed reactive hyperplasia. These cases suggest that some cases are so complex that avid 18F-FDG uptake in the mediastinal lymph nodes in the patients with pathologically diagnosed lung cancer should be carefully analyzed based on individual patients’ clinical background.
PMCID: PMC4270568  PMID: 25550877
FDG; PET-CT; hypermetabolic; mediastinal lymph nodes; lung cancer
5.  Contribution of T Cell Subsets to the Pathophysiology of Pneumocystis-Related Immunorestitution Disease 
Immune-mediated lung injury is an important component of Pneumocystis pneumonia (PcP)-related immunorestitution disease (IRD). However, the individual contribution of CD4+ and CD8+ T cells to the pathophysiology of IRD remains undetermined. Therefore, IRD was modeled in severe combined immunodeficient mice, and specific T cell depletion was used to determine how T cell subsets interact to affect the nature and severity of disease. CD4+ cells were more abundant than CD8+ cells during the acute stage of IRD that coincided with impaired pulmonary physiology and organism clearance. Conversely, CD8+ cells were more abundant during the resolution phase following P. carinii clearance. Depletion of CD4+ T cells protected mice from the acute pathophysiology of IRD. However, these mice could not clear the infection and developed severe PcP at later time points when a pathological CD8+ T cell response was observed. In contrast, mice depleted of CD8+ T cells efficiently cleared the infection, but developed more severe disease, an increased frequency of IFN-γ-producing CD4+ cells, and a prolonged CD4+ T cell response than mice with both CD4+ and CD8+ cells. These data suggest that CD4+ T cells mediate the acute respiratory disease associated with IRD. In contrast, CD8+ T cells contributed to neither lung injury nor organism clearance when CD4+ cells were present, but instead served to modulate CD4 function. In the absence of CD4+ cells, CD8+ T cells produced a non-protective, pathological immune response. These data suggest that the interplay of CD4+ and CD8+ T cells affects the ultimate outcome of PcP-related IRD.
PMCID: PMC4161614  PMID: 16891394
Inflammation; Pulmonary Physiology; AIDS; T cells; Pneumocystis
6.  Activation of Extracellular Signal-Regulated Kinase 5 Reduces Cardiac Apoptosis and Dysfunction via Inhibition of a Phosphodiesterase 3A/Inducible cAMP Early Repressor Feedback Loop 
Circulation research  2007;100(4):510-519.
Substantial evidence suggests that the progressive loss of cardiomyocytes caused by apoptosis significantly contributes to the development of heart failure. β-Adrenergic receptor activation and subsequent persistent phosphodiesterase 3A (PDE3A) downregulation and concomitant inducible cAMP early repressor (ICER) upregulation (PDE3A/ICER feedback loop) has been proposed to play a key role in the pathogenesis of cardiomyocyte apoptosis. In contrast, insulin-like growth factor-1 can activate cell survival pathways, providing protection against cell death and restoring muscle function. In this study, we found that insulin-like growth factor-1 activates extracellular signal-regulated kinase 5 (ERK5) and inhibits PDE3A/ICER feedback loop. Insulin-like growth factor-1 normalized isoproterenol-mediated PDE3A downregulation and ICER upregulation via ERK5/MEF2 activation, and also inhibited isoproterenol-induced myocyte apoptosis. To determine the physiological relevance of ERK5 activation in regulating PDE3A/ICER feedback loop, we investigated the PDE3A/ICER expression and cardiomyocyte apoptosis in transgenic mice with cardiac specific expression of a constitutively active form of mitogen-activated protein (MAP)/extracellular signal-regulated protein kinase (ERK) kinase 5α (MEK5α) (CA-MEK5α-Tg). In wild-type mice, pressure overload– or doxorubicin-induced significant reduction of PDE3A expression and subsequent ICER induction. Cardiac specific expression of CA-MEK5α rescued pressure overload– or doxorubicin-mediated PDE3A downregulation and ICER upregulation and inhibited myocyte apoptosis as well as subsequent cardiac dysfunction in vivo. These data suggest that preventing the feedback loop of PDE3A/ICER by ERK5 activation could inhibit progression of myocyte apoptosis as well as cardiac dysfunction. These data suggest a new therapeutic paradigm for end stage of heart failure by inhibiting the PDE3A/ICER feedback loop via activating ERK5.
PMCID: PMC4115673  PMID: 17272811
ERK5; phosphodiesterase 3; inducible cAMP early repressor; heart failure; insulin-like growth factor-1
7.  A Crucial Role for p90RSK-Mediated Reduction of ERK5 Transcriptional Activity in Endothelial Dysfunction and Atherosclerosis 
Circulation  2012;127(4):486-499.
Diabetes mellitus (DM) is a major risk factor for cardiovascular mortality by increasing endothelial cell (EC) dysfunction and subsequently accelerating atherosclerosis. Extracellular-signal regulated kinase 5 (ERK5) is activated by steady laminar flow and regulates EC function by increasing eNOS expression and inhibiting EC inflammation. However, the role and regulatory mechanisms of ERK5 in EC dysfunction and atherosclerosis are poorly understood. Here, we report the critical role of the p90 ribosomal S6 kinase (p90RSK)/ERK5 complex in EC dysfunction in DM and atherosclerosis.
Methods and Results
Inducible EC-specific ERK5 knockout (ERK5-EKO) mice showed increased leukocyte rolling and impaired vessel reactivity. To examine the role of endothelial ERK5 in atherosclerosis, we used inducible ERK5-EKO-LDLR−/− mice and observed increased plaque formation. When activated, p90RSK associated with ERK5, and this association inhibited ERK5 transcriptional activity and up-regulated VCAM-1 expression. In addition, p90RSK directly phosphorylated ERK5 S496 and reduced eNOS expression. p90RSK activity was increased in diabetic mouse vessels, and FMK-MEA, a specific p90RSK inhibitor, ameliorated EC-leukocyte recruitment and diminished vascular reactivity in DM mice. Interestingly, in ERK-EKO mice, increased leukocyte rolling and impaired vessel reactivity were resistant to the beneficial effects of FMK-MEA, suggesting a critical role for endothelial ERK5 in mediating the salutary effects of FMK-MEA on endothelial dysfunction. FMK-MEA also inhibited atherosclerosis formation in ApoE−/− mice.
Our study highlights the importance of the p90RSK/ERK5 module as a critical mediator of EC dysfunction in DM and atherosclerosis formation, thus revealing a potential new target for therapeutic intervention.
PMCID: PMC3574639  PMID: 23243209
Diabetes mellitus; endothelial dysfunction; p90RSK; ERK5; signal transduction
8.  Canonical Wnt/β-catenin signaling in epicardial fibrosis of failed pediatric heart allografts with diastolic dysfunction 
Failed pediatric heart allografts with diastolic dysfunction exhibit severe epicardial fibrosis. The molecular mechanism underlying this process is poorly understood. Canonical Wnt/β-catenin signaling plays an important role in epithelial-mesenchymal transition and is implicated in fibrosing diseases. In this study, we tested the hypothesis that canonical Wnt/β-catenin signaling is activated in epicardial fibrosis of end-stage dysfunctional pediatric allografts.
Fourteen explanted heart grafts of 12 patients who had undergone 14 heart transplantations were used for immunohistochemical staining of β-catenin and its nuclear binding partners, T-cell factor/lymphoid enhancer factor (TCF/LEF) family transcriptional factors. Fourteen age-matched native hearts from patients who undergone first heart transplantation without evidence of epicardial fibrosis were used as controls.
Results and Conclusions
Epicardial fibroblasts from explanted allografts demonstrated nuclear accumulation of β-catenin. These cells also showed nuclear positivity for TCF-4. No TCF-3 expression was present in the epicardium. TCF-1 and LEF-1 were observed in lymphocytes, but not in other cell types of the epicardium. These findings suggest an association between canonical Wnt/beta-catenin signaling and epicardial fibrosis of failed pediatric heart allografts. Should activation of this pathway be shown to be causal to epicardial fibrosis in this setting, then inhibition of this pathway may help to prevent this devastating process.
PMCID: PMC3427707  PMID: 22475572
Heart transplant; Pediatric; Epicardium; Fibrosis; Allograft; Heart failure; Wnt signaling; LEF/TCF; β-Catenin
9.  High frequency of coexpression of maspin with p63 and p53 in squamous cell carcinoma but not in adenocarcinoma of the lung 
Maspin, a member of the serpin family of protease inhibitors, has been shown to inhibit tumor growth and suppress metastasis in several malignancies, including lung cancer. Previous studies have reported that p63 and p53 control maspin expression by transactivating the promoter. The present study analyzed immunohistochemical studies to determine the expression and coexpression patterns of maspin, p63 and p53 in non-small cell lung carcinoma, specifically squamous cell carcinoma and adenocarcinoma. The results showed that 83/86 cases (96.5%) of squamous cell carcinoma and 82/161 cases (50.9%) of adenocarcinoma included in this study were positive for maspin. There were 79/86 cases (91.9%) of squamous cell carcinoma and 16/161 cases (9.9%) of adenocarcinoma with positive expression for p63. In addition, 77/86 cases (89.5%) of squamous cell carcinoma and 99/161 cases (61.5%) of adenocarcinoma were positive for p53. Maspin, p63 and p53 expression were each significantly higher in squamous cell carcinoma than adenocarcinoma. Squamous cell carcinomas more highly coexpress maspin and p63, as well as maspin and p53, when compared with adenocarcinomas. The high frequency of coexpression of maspin and p63, as well as maspin and p53, in squamous cell carcinoma, suggests that p63 and p53 may be involved in the pathway to control maspin expression. Therapeutic targeting on maspin, p63 and p53 molecules might be beneficial in the management of patients with squamous cell carcinomas of the lung in the future.
PMCID: PMC3816825  PMID: 24228118
Maspin; p63; p53; lung; adenocarcinoma; squamous cell carcinoma
10.  Immunohistochemical detection of mutations in the epidermal growth factor receptor gene in lung adenocarcinomas using mutation-specific antibodies 
Diagnostic Pathology  2013;8:27.
The recent development of antibodies specific for the major hotspot mutations in the epidermal growth factor receptor (EGFR), L858R and E746_A750del, may provide an opportunity to use immunohistochemistry (IHC) as a screening test for EGFR gene mutations. This study was designed to optimize the IHC protocol and the criteria for interpretation of the results using DNA sequencing as the gold-standard.
Tumor sections from fifty lung adenocarcinoma specimens from Chinese patients were immunostained using L858R and E746_A750del-specific antibodies using three different antigen retrieval solutions, and the results were evaluated using three different sets of criteria. The same specimens were used for DNA purification and analysis of EGFR gene mutations.
In this study the optimal buffer for antigen retrieval was EDTA (pH 8.0), and the optimal scoring method was to call positive results when there was moderate to strong staining of membrane and/or cytoplasm in >10% of the tumor cells. Using the optimized protocol, L858R-specific IHC showed a sensitivity of 81% and a specificity of 97%, and E746_A750del-specific IHC showed a sensitivity of 59% and a specificity of 100%, both compared with direct DNA analysis. Additionally, the mutant proteins as assessed by IHC showed a more homogeneous than heterogeneous pattern of expression.
Our data demonstrate that mutation-specific IHC, using optimized procedures, is a reliable prescreening test for detecting EGFR mutations in lung adenocarcinoma.
Virtual Slides
The virtual slide(s) for this article can be found here:
PMCID: PMC3635899  PMID: 23419122
Lung adenocarcinoma; Epidermal growth factor receptor; Mutation; Immunohistochemistry
11.  Expression of glypican-3 in undifferentiated embryonal sarcoma and mesenchymal hamartoma of the liver☆ 
Human pathology  2011;43(5):695-701.
Glypican-3 (GPC3) is an oncofetal protein that has been demonstrated to be a useful diagnostic immunomarker for hepatocellular carcinoma and hepatoblastoma. Its expression in mesenchymal tumors of the liver, particularly undifferentiated embryonal sarcoma (UES) and mesenchymal hamartoma (MH), has not been investigated. In this study, a total of 24 UESs and 18 MHs were immunohistochemically stained for GPC3 expression. The results showed cytoplasmic staining for GPC3 in 14 (58%) UESs, of which 6 exhibited diffuse immunoreactivity and the remaining 8 showed focal positivity. The patients with GPC3-positive UES tended to be younger (mean 18 years; median 11 years) than those with GPC3-negative tumors (mean 39.4 years; median 27 years), although the difference did not reach statistical significance (P = .06). Eight MHs also exhibited GPC3 immunoreactivity (44%; 4 diffuse and 4 focal). Positive staining in all 8 cases was primarily seen in entrapped nonlesional hepatocytes with a canalicular and cytoplasmic staining pattern. In only 4 cases (22%) was GPC3 immunoreactivity also observed in the mesenchymal component. The patients with positive staining also tended to be younger (mean 2.6 years; median 1.1 years) compared with those with negative staining (mean 16.3 years; median 4.5 years), but the difference was not statistically significant (P = .15). Our data demonstrate that GPC3 is expressed in a subset of UES and MH of the liver. Caution should thus be exercised when evaluating a GPC3-expressing hepatic neoplasm, particularly on a needle biopsy when the differential diagnosis includes poorly differentiated hepatocellular carcinoma or hepatoblastoma.
PMCID: PMC3568522  PMID: 21937079
Glypican-3; Liver; Undifferentiated embryonal sarcoma; Mesenchymal hamartoma; Immunohistochemistry
12.  Yin Yang 1 Is a Novel Regulator of Pulmonary Fibrosis 
Rationale: The differentiation of fibroblasts into myofibroblasts is a cardinal feature of idiopathic pulmonary fibrosis (IPF). The transcription factor Yin Yang 1 (YY1) plays a role in the proliferation and differentiation of diverse cell types, but its role in fibrotic lung diseases is not known.
Objectives: To elucidate the mechanism by which YY1 regulates fibroblast differentiation and lung fibrosis.
Methods: Lung fibroblasts were cultured with transforming growth factor (TGF)-β or tumor necrosis factor-α. Nuclear factor (NF)-κB, YY1, and α-smooth muscle actin (SMA) were determined in protein, mRNA, and promoter reporter level. Lung fibroblasts and lung fibrosis were assessed in a partial YY1-deficient mouse and a YY1f/f conditional knockout mouse after being exposed to silica or bleomycin.
Measurements and Main Results: TGF-β and tumor necrosis factor-α up-regulated YY1 expression in lung fibroblasts. TGF-β–induced YY1 expression was dramatically decreased by an inhibitor of NF-κB, which blocked I-κB degradation. YY1 is significantly overexpressed in both human IPF and murine models of lung fibrosis, including in the aggregated pulmonary fibroblasts of fibrotic foci. Furthermore, the mechanism of fibrogenesis is that YY1 can up-regulate α-SMA expression in pulmonary fibroblasts. YY1-deficient (YY1+/−) mice were significantly protected from lung fibrosis, which was associated with attenuated α-SMA and collagen expression. Finally, decreasing YY1 expression through instilled adenovirus-cre in floxed-YY1f/f mice reduced lung fibrosis.
Conclusions: YY1 is overexpressed in fibroblasts in both human IPF and murine models in a NF-κB–dependent manner, and YY1 regulates fibrogenesis at least in part by increasing α-SMA and collagen expression. Decreasing YY1 expression may provide a new therapeutic strategy for pulmonary fibrosis.
PMCID: PMC3136995  PMID: 21169469
nuclear factor-κB; α-smooth muscle actin; idiopathic pulmonary fibrosis
13.  Reactive Oxygen Species Suppress Cardiac NaV1.5 Expression through Foxo1 
PLoS ONE  2012;7(2):e32738.
NaV1.5 is a cardiac voltage-gated Na+ channel αsubunit and is encoded by the SCN5a gene. The activity of this channel determines cardiac depolarization and electrical conduction. Channel defects, including mutations and decrease of channel protein levels, have been linked to the development of cardiac arrhythmias. The molecular mechanisms underlying the regulation of NaV1.5 expression are largely unknown. Forkhead box O (Foxo) proteins are transcriptional factors that bind the consensus DNA sequences in their target gene promoters and regulate the expression of these genes. Comparative analysis revealed conserved DNA sequences, 5′-CAAAACA-3′ (insulin responsive element, IRE), in rat, mouse and human SCN5a promoters with the latter two containing two overlapping Foxo protein binding IREs, 5′-CAAAACAAAACA-3′. This finding led us to hypothesize that Foxo1 regulates NaV1.5 expression by directly binding the SCN5a promoter and affecting its transcriptional activity. In the present study, we determined whether Foxo1 regulates NaV1.5 expression at the transcriptional level and also defined the role of Foxo1 in hydrogen peroxide (H2O2)-mediated NaV1.5 suppression in HL-1 cardiomyocytes using chromatin immunoprecipitation (ChIP), constitutively nuclear Foxo1 expression, and RNAi Foxo1 knockdown as well as whole cell voltage-clamp recordings. ChIP with anti-Foxo1 antibody and follow-up semi-quantitative PCR with primers flanking Foxo1 binding sites in the proximal SCN5a promoter region clearly demonstrated enrichment of DNA, confirming Foxo1 recruitment to this consensus sequence. Foxo1 mutant (T24A/S319A-GFP, Foxo1-AA-GFP) was retained in nuclei, leading to a decrease of NaV1.5 expression and Na+ current, while silencing of Foxo1 expression by RNAi resulted in the augmentation of NaV1.5 expression. H2O2 significantly reduced NaV1.5 expression by promoting Foxo1 nuclear localization and this reduction was prevented by RNAi silencing Foxo1 expression. These studies indicate that Foxo1 negatively regulates NaV1.5 expression in cardiomyocytes and reactive oxygen species suppress NaV1.5 expression through Foxo1.
PMCID: PMC3293505  PMID: 22400069
14.  Role of Ca2+/calmodulin-stimulated cyclic nucleotide phosphodiesterase 1 in mediating cardiomyocyte hypertrophy 
Circulation research  2009;105(10):956-964.
Cyclic nucleotide phosphodiesterases (PDE) through the degradation of second messenger cyclic guanosine monophosphate (cGMP) play critical roles in maintaining cardiomyocyte homeostasis. Ca2+/CaM-activated cGMP-hydrolyzing PDE1 family may play a pivotal role in balancing intracellular Ca2+/CaM and cGMP signaling, however its function in cardiomyocytes is unknown.
Herein we investigate the role of Ca2+/CaM-stimulated PDE1 in regulating pathological cardiomyocyte hypertrophy in neonatal and adult rat ventricular myocytes (NRVM and ARVM) and in the heart in vivo.
Methods and Results
Inhibition of PDE1 activity using a PDE1 selective inhibitor IC86340 or downregulation of PDE1A using siRNA prevented phenylephrine (PE) induced pathological myocyte hypertrophy and hypertrophic marker expression in neonatal (NRVM) and adult (ARVM) rat ventricular myocytes. Importantly, administration of the PDE1 inhibitor IC86340 attenuated cardiac hypertrophy induced by chronic ISO infusion in vivo. Both PDE1A and PDE1C mRNA and protein were detected in human hearts, however PDE1A expression was conserved in rodent hearts. Moreover, PDE1A expression was significantly upregulated in vivo in the heart and myocytes from various pathological hypertrophy animal models and in vitro in isolated NRVM and ARVM treated with neurohumoral stimuli such as angiotensin II (Ang II) and ISO. Further, PDE1A plays a critical role in PE-induced reduction of intracellular cGMP and PKG activity, and thereby cardiomyocyte hypertrophy in vitro.
These results elucidate a novel role for Ca2+/CaM-stimulated PDE1, particularly PDE1A, in regulating pathological cardiomyocyte hypertrophy via a cGMP/PKG-dependent mechanism, thereby demonstrating Ca2+ and cGMP signaling cross-talk during cardiac hypertrophy.
PMCID: PMC2803071  PMID: 19797176
phosphodiesterase; cGMP; cardiomyocyte; cardiac hypertrophy
15.  Reactive Oxygen Species-Induced Activation of p90 Ribosomal S6 Kinase Prolongs Cardiac Repolarization via Inhibiting Outward K+ Channel Activity 
Circulation research  2008;103(3):269-278.
p90 ribosomal S6 kinase (p90RSK) is activated in cardiomyopathies caused by conditions such as ischemia/reperfusion injury (I/R) and diabetes mellitus (DM), in which prolongation of cardiac repolarization and frequent arrhythmias are common. Molecular mechanisms underlying the electrical remodeling in cardiac diseases are largely unknown. In the present study, we determined the role of p90RSK activation in the modulation of voltage-gated K+ channel activity determining cardiac repolarization. Mice with increased cardiac p90RSK activity due to transgenic expression of p90RSK (p90RSK-Tg) had prolongation of QT intervals and of ventricular myocyte action potential durations. Fast transient outward K+ current (Ito,f), slow delayed outward K+ current (IK,slow) and steady-state K+ current (ISS) were significantly decreased in p90RSK-Tg mouse ventricular myocytes. mRNA levels of Kv4.3, Kv4.2, Kv1.5, Kv2.1 and KChIP2 from ventricles between p90RSK-Tg and non-transgenic littermate control mice were similar, as assessed by quantitative RT-PCR, indicating that p90RSK regulates voltage-gated K+ channels through post-translational modification. Kv4.3- and Kv1.5- rather than Kv4.2- and Kv2.1-encoded channels in HEK 293 cells were inhibited by p90RSK. In vitro phosphorylation analysis showed that Kv4.3 was phosphorylated by p90RSK at two conserved sites, Ser516 and Ser550. p90RSK expression significantly inhibited Kv4.3-, and Kv4.3 and KChIP2-encoded channel activities in HEK 293 cells while p90RSK’s effects were blocked by amino acid mutation(s) at phosphorylation site(s) in Kv4.3. Hydrogen peroxide (H2O2), a mediator of induced cardiac p90RSK activation in I/R and DM, had effects similar to those of p90RSK on Kv4.3- or Kv4.3 and KChIP2-encoded channels. Fluoromethylketone, a specific p90RSK inhibitor, abolished H2O2 effects. These findings indicate that p90RSK activation is critical for reactive oxygen species-mediated inhibition of voltage-gatedK+ channel activity and leads to prolongation of cardiac repolarization.
PMCID: PMC2631445  PMID: 18599872
Reactive oxygen species; hydrogen peroxide; p90RSK; phosphorylation; voltage-gated outward K+ currents; cardiac repolarization; arrhythmias
16.  Tumor Suppressor CYLD Acts as a Negative Regulator for Non-Typeable Haemophilus influenza-Induced Inflammation in the Middle Ear and Lung of Mice 
PLoS ONE  2007;2(10):e1032.
Non-typeable Haemophilus influenza (NTHi) is an important human pathogen causing respiratory tract infections in both adults and children. NTHi infections are characterized by inflammation, which is mainly mediated by nuclear transcription factor kappaB (NF-κB)-dependent production of inflammatory mediators. The deubiquitinating enzyme cylindromatosis (CYLD), loss of which was originally reported to cause a benign human syndrome called cylindromatosis, has been identified as a key negative regulator for NF-κB in vitro. However, little is known about the role of CYLD in bacteria-induced inflammation in vivo. Here, we provided direct evidence for the negative role of CYLD in NTHi-induced inflammation of the mice in vivo. Our data demonstrated that CYLD is induced by NTHi in the middle ear and lung of mice. NTHi-induced CYLD, in turn, negatively regulates NTHi-induced NF-κB activation through deubiquitinating TRAF6 and 7 and down-regulates inflammation. Our data thus indicate that CYLD acts as a negative regulator for NF-κB-dependent inflammation in vivo, hence protecting the host against detrimental inflammatory response to NTHi infection.
PMCID: PMC2001183  PMID: 17925880
17.  Four Kinetically Distinct Depolarization-activated K+ Currents in Adult Mouse Ventricular Myocytes  
The Journal of General Physiology  1999;113(5):661-678.
In the experiments here, the time- and voltage-dependent properties of the Ca2+-independent, depolarization-activated K+ currents in adult mouse ventricular myocytes were characterized in detail. In the majority (65 of 72, ≈ 90%) of cells dispersed from the ventricles, analysis of the decay phases of the outward currents revealed three distinct K+ current components: a rapidly inactivating, transient outward K+ current, Ito,f (mean ± SEM τdecay = 85 ± 2 ms); a slowly (mean ± SEM τdecay = 1,162 ± 29 ms) inactivating K+ current, IK,slow; and a non inactivating, steady state current, Iss. In a small subset (7 of 72, ≈ 10%) of cells, Ito,f was absent and a slowly inactivating (mean ± SEM τdecay = 196 ± 7 ms) transient outward current, referred to as Ito,s, was identified; the densities and properties of IK,slow and Iss in Ito,s-expressing cells are indistinguishable from the corresponding currents in cells with Ito,f. Microdissection techniques were used to remove tissue pieces from the left ventricular apex and from the ventricular septum to allow the hypothesis that there are regional differences in Ito,f and Ito,s expression to be tested directly. Electrophysiological recordings revealed that all cells isolated from the apex express Ito,f (n = 35); Ito,s is not detected in these cells (n = 35). In the septum, by contrast, all of the cells express Ito,s (n = 28) and in the majority (22 of 28, 80%) of cells, Ito,f is also present. The density of Ito,f (mean ± SEM at +40 mV = 6.8 ± 0.5 pA/pF, n = 22) in septum cells, however, is significantly (P < 0.001) lower than Ito,f density in cells from the apex (mean ± SEM at +40 mV = 34.6 ± 2.6 pA/pF, n = 35). In addition to differences in inactivation kinetics, Ito,f, Ito,s, and IK,slow display distinct rates of recovery (from inactivation), as well as differential sensitivities to 4-aminopyridine (4-AP), tetraethylammonium (TEA), and Heteropoda toxin-3. IK,slow, for example, is blocked selectively by low (10–50 μM) concentrations of 4-AP and by (≥25 mM) TEA. Although both Ito,f and Ito,s are blocked by high (>100 μM) 4-AP concentrations and are relatively insensitive to TEA, Ito,f is selectively blocked by nanomolar concentrations of Heteropoda toxin-3, and Ito,s (as well as IK,slow and Iss) is unaffected. Iss is partially blocked by high concentrations of 4-AP or TEA. The functional implications of the distinct properties and expression patterns of Ito,f and Ito,s, as well as the likely molecular correlates of these (and the IK,slow and Iss) currents, are discussed.
PMCID: PMC2222908  PMID: 10228181
Ito,f ; Ito,s ; transient outward currents; delayed rectifier; transgenic mice
18.  CYLD negatively regulates transforming growth factor-β-signalling via deubiquitinating Akt 
Nature Communications  2012;3:771-.
Lung injury, whether induced by infection or caustic chemicals, initiates a series of complex wound-healing responses. If uncontrolled, these responses may lead to fibrotic lung diseases and loss of function. Thus, resolution of lung injury must be tightly regulated. The key regulatory proteins required for tightly controlling the resolution of lung injury have yet to be identified. Here we show that loss of deubiquitinase CYLD led to the development of lung fibrosis in mice after infection with Streptococcus pneumoniae. CYLD inhibited transforming growth factor-β-signalling and prevented lung fibrosis by decreasing the stability of Smad3 in an E3 ligase carboxy terminus of Hsc70-interacting protein-dependent manner. Moreover, CYLD decreases Smad3 stability by deubiquitinating K63-polyubiquitinated Akt. Together, our results unveil a role for CYLD in tightly regulating the resolution of lung injury and preventing fibrosis by deubiquitinating Akt. These studies may help develop new therapeutic strategies for preventing lung fibrosis.
Lung injury initiates a series of wound-healing responses, which if unregulated, can lead to fibrosis. Li et al. show that the deubquitinase CYLD has a key role in the prevention of fibrosis by inhibiting transforming growth factor β-signalling through the direct deubiquitination of the protein kinase Akt.
PMCID: PMC3337989  PMID: 22491319

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